EP0047485A2 - Process for the propagation of protozoal and their use - Google Patents

Process for the propagation of protozoal and their use Download PDF

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Publication number
EP0047485A2
EP0047485A2 EP81106855A EP81106855A EP0047485A2 EP 0047485 A2 EP0047485 A2 EP 0047485A2 EP 81106855 A EP81106855 A EP 81106855A EP 81106855 A EP81106855 A EP 81106855A EP 0047485 A2 EP0047485 A2 EP 0047485A2
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Prior art keywords
protozoa
serum
medium
erythrocytes
plasmodia
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German (de)
French (fr)
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EP0047485A3 (en
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Burkhard Dr. Enders
Hans Heinrich Hahn
Günter Neunziger
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Siemens Healthcare Diagnostics GmbH Germany
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Behringwerke AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to a method for the propagation of protozoa, especially Plasmodia, in particular Plasmodium falciparum, in a medium containing serum and optionally host cells, to which nutrients are added by dialysis and from which metabolic products are simultaneously removed.
  • the application accordingly relates to a process for the propagation of protozoa, in particular Plasmodia, in particular Plasmodium falciparum, characterized in that a suspension which contains at least protozoa and serum and, if appropriate, host cells is separated from a solution by a membrane which is permeable only to low molecular weight substances, which contains low-molecular nutrients necessary for the proliferation of protozoa and, on the other hand, can absorb harmful low-molecular metabolic products.
  • Plasmodium falciparum is the causative agent of malaria, and methods of cultivating it are of great importance in the search for means to combat this common disease.
  • the process according to the invention consists in that a suspension of protozoa and, if appropriate, host cells in a defined medium contains the high-molecular substances (e.g. serum proteins) necessary for cultivation and, if appropriate, further constituents and is separated from one by a membrane which only allows low-molecular substances to pass through Solution that contains only low-molecular substances necessary for cultivation.
  • a suspension of protozoa and, if appropriate, host cells in a defined medium contains the high-molecular substances (e.g. serum proteins) necessary for cultivation and, if appropriate, further constituents and is separated from one by a membrane which only allows low-molecular substances to pass through Solution that contains only low-molecular substances necessary for cultivation.
  • Possible protozoa are: plasmodia, piroplasmas, trypanosomes, leishmanias, trichomonads, amoebas, toxoplasmas.
  • the method is particularly suitable for the plasmodia: P. falciparum, P. fragile, P. malariae, P. knowlesi, P. gallinaceum, in particular P. falciparum.
  • the low molecular weight portions of serum and, if appropriate, culture media containing host cells, which are suitable for the cultivation of protozoa, are known. These are essentially amino acids, vitamins, peptone and physiologically acceptable salts.
  • a medium commercially available under the name RPMI 1640 which is described below, can be used.
  • RPMI 1640 which is described below, can be used.
  • suitably modified "Medium 199" (Haynes et al., 1976).
  • the preferred serum used is: human serum A 1 , Rh; Human serum O.
  • erythrocytes are used, the following can be used: human erythrocytes A, Rh; Human erythrocytes 0.
  • lactate lactic acid enriched by the parasite metabolism, which normally causes a growth-inhibiting local pH change in the medium, is removed by the dialysis method, whereby more favorable growth conditions are achieved (pH stability, increase in the proportion of erythrocytes offered and thereby higher yield on malaria parasites, saving on unused high molecular serum components):
  • Plasmodium falciparum are suitable for the production of an anti-parasitic (malaria) vaccine.
  • the Plasmodium falciparum merozoites harvested after growth are concentrated, attenuated (chemically or physically) and brought into a vaccine-compatible form.
  • the invention accordingly relates in particular to the use of the parasites produced by the process according to the invention as an essential active ingredient in a vaccine or in a diagnostic agent.

Abstract

A process for the propagation of protozoa is described in which nutrients are added and metabolic products are removed by means of dialysis. The protozoa thus obtained can be used in a vaccine or diagnostic.

Description

Die Erfindung betrifft ein Verfahren zur Vermehrung von Protozoen, besonders von Plasmodien, insbesondere Plasmodium falciparum, in einem Serum und gegebenenfalls Wirtszellen enthaltenden Medium, welchem mittels Dialyse Nährstoffe zugeführt und aus welchem gleichzeitig Stoffwechselprodukte entfernt werden.The invention relates to a method for the propagation of protozoa, especially Plasmodia, in particular Plasmodium falciparum, in a medium containing serum and optionally host cells, to which nutrients are added by dialysis and from which metabolic products are simultaneously removed.

Es sind Verfahren zur Vermehrung von Plasmodien in Erythrozyten bekannt, in welchen das vollständige serumhaltige Nährmedium kontinuierlich oder automatisch periodisch erneuert wird. So wird in dem von TRAGER, W., J.Protozooi. 26 (1), 1979, S. 125-129,beschriebenen Verfahren das Kulturmedium kontinuierlich über die Suspension von Plasmodien und Erythrozyten geführt. Das von JENSEN, J.B., TRAGER, W. and DOHERTY, J. in Experimental Parasitology 48, 1979, S. 36-41 beschriebene Verfahren arbeitet mit einer Vorrichtung, die es gestattet, das Kulturmedium automatisch periodisch von der Suspension von Plasmodien und Erythrozyten abzudekantieren und frisches Kulturmedium zuzuführen.Methods for multiplying plasmodia in erythrocytes are known in which the complete serum-containing nutrient medium is continuously or automatically renewed periodically. So in the by TRAGER, W., J.Protozooi. 26 (1), 1979, pp. 125-129, described the culture medium continuously over the suspension of plasmodia and erythrocytes. The method described by JENSEN, JB, TRAGER, W. and DOHERTY, J. in Experimental Parasitology 48, 1979, pp. 36-41 works with a device which allows the culture medium to be automatically periodically decanted from the suspension of plasmodia and erythrocytes and supply fresh culture medium.

Diese Verfahren haben den Nachteil, daß beim Wechsel des mit inhibierenden Stoffwechselprodukten angereicherten Kulturmediums auch wertvolles noch nicht verbrauchtes Humanserum sowie Protozoen (freie Merozoiten) und Wirtszellen (Erythrozyten) verlorengehen. Außerdem sind diese Methoden nicht für eine Vermehrung in größerem Maßstab geeignet.These processes have the disadvantage that when the culture medium enriched with inhibitory metabolic products is changed, valuable human serum which has not yet been consumed, as well as protozoa (free merozoites) and host cells (erythrocytes) are lost. In addition, these methods are not suitable for large-scale propagation.

Es stellte sich daher die Aufgabe, ein Verfahren zu finden, mittels welchem Protozoen, wie beispielsweise für eine Impfstoffherstellung erforderlich, in größeren Mengen und hoher Ausbeute in wirtschaftlicher Weise vermehrt werden können.It was therefore the task of finding a process by means of which protozoa, as required, for example, for vaccine production, can be multiplied in large quantities and in a high yield in an economical manner.

Diese Aufgabe konnte dadurch gelöst werden, daß überraschend gefunden wurde, daß sich Protozoen, besonders Plasmodien, insbesondere Plasmodium falciparum, mit guter Ausbeute vermehren lassen, wenn man die Serum enthaltende Kultursuspension von Protozoen und gegebenenfalls Wirtszellen durch eine Dialyse-Membran trennt von einer serumfreien Lösung, welche die niedermolekularen zur Vermehrung nötigen Nährstoffe enthält. Diese Lösung kann gewechselt werden, ohne daß Protozoen, Wirtszellen oder Serum verlorengehen. Außerdem werden die in der Kultur angereicherten, das Wachstum der Protozoen hemmenden, niedermolekularen metabolischen Produkte, vornehmlich Lactat, durch das Dialyseverfahren entfernt.This object was achieved in that it was surprisingly found that protozoa, especially Plasmodia, in particular Plasmodium falciparum, can be reproduced with good yield if the culture suspension of protozoa and, if appropriate, host cells containing serum is separated from a serum-free solution by a dialysis membrane , which contains the low-molecular nutrients necessary for reproduction. This solution can be changed without losing protozoa, host cells or serum. In addition, the culture-enriched, low-molecular metabolic products, mainly lactate, which inhibit the growth of protozoa, are removed by the dialysis process.

Gegenstand der Anmeldung ist demnach ein Verfahren zur Vermehrung von Protozoen, besonders Plasmodien, insbesondere Plasmodium falciparum, dadurch gekennzeichnet, daß eine Suspension, die mindestens Protozoen und Serum sowie gegebenenfalls Wirtszellen enthält, durch eine lediglich für niedermolekulare Stoffe durchlässige Membran getrennt ist von einer Lösung, die niedermolekulare für die Vermehrung der Protozoen nötige Nährstoffe enthält und andererseits schädliche niedermolekulare Stoffwechselprodukte aufnehmen kann.The application accordingly relates to a process for the propagation of protozoa, in particular Plasmodia, in particular Plasmodium falciparum, characterized in that a suspension which contains at least protozoa and serum and, if appropriate, host cells is separated from a solution by a membrane which is permeable only to low molecular weight substances, which contains low-molecular nutrients necessary for the proliferation of protozoa and, on the other hand, can absorb harmful low-molecular metabolic products.

Plasmodium falciparum ist der Erreger der Malaria, und Verfahren zu seiner Kultivierung haben eine große Bedeutung für die Suche nach Mitteln zur Bekämpfung dieser verbreiteten Krankheit.Plasmodium falciparum is the causative agent of malaria, and methods of cultivating it are of great importance in the search for means to combat this common disease.

Es ist bekannt, daß zum Wachstum und zur Vermehrung von Plasmodien ein Kulturmedium erforderlich ist, das Humanserum und Erythrozyten der mit dem Serum kompatiblen Blutgruppe enthält, und das von Zeit zu Zeit erneuert werden muß. Es war jedoch nicht bekannt, daß nicht das komplette Kulturmedium ausgetauscht werden muß, sondern daß es überraschenderweise genügt, für eine Zu- und Abführung der niedermolekularen Anteile (essentielle Aminosäuren, inhibierende Stoffwechselprodukte) zu sorgen. Das erfindungsgemäße Verfahren besteht darin, daß eine Suspension von Protozoen und gegebenenfalls Wirtszellen in einem definierten Medium die für die Kultivierung nötigen hochmolekularen Stoffe (z.B. Serumproteine) sowie gegebenenfalls weitere Bestandteile enthält und durch eine Membran, die nur niedermolekulare Stoffe passieren läßt, getrennt ist von einer Lösung, welche nur niedermolekulare für die Kultivierung nötige Stoffe enthält.It is known that a culture medium which contains human serum and erythrocytes of the blood group compatible with the serum and which has to be renewed from time to time is required for the growth and multiplication of plasmodia. However, it was not known that the complete culture medium does not have to be replaced, but that it is surprisingly sufficient to ensure that the low molecular weight portions (essential amino acids, inhibiting metabolic products) are supplied and removed. The process according to the invention consists in that a suspension of protozoa and, if appropriate, host cells in a defined medium contains the high-molecular substances (e.g. serum proteins) necessary for cultivation and, if appropriate, further constituents and is separated from one by a membrane which only allows low-molecular substances to pass through Solution that contains only low-molecular substances necessary for cultivation.

Als Protozoen kommen in Frage: Plasmodien, Piroplasmen, Trypanosomen, Leishmanien, Trichomonaden, Amoeben, Toxoplasmen.Possible protozoa are: plasmodia, piroplasmas, trypanosomes, leishmanias, trichomonads, amoebas, toxoplasmas.

Besonders geeignet ist das Verfahren für die Plasmodien: P. falciparum, P. fragile, P. malariae, P. knowlesi, P. gallinaceum, insbesondere P. falciparum.The method is particularly suitable for the plasmodia: P. falciparum, P. fragile, P. malariae, P. knowlesi, P. gallinaceum, in particular P. falciparum.

Die niedermolekularen Anteile von Serum und gegebenenfalls Wirtszellen enthaltenden Nährmedien, die für die Kultivierung von Protozoen geeignet sind, sind bekannt. Im wesentlichen sind dies Aminosäuren, Vitamine, Pepton und physiologisch verträgliche Salze. Für Plasmodium falciparum ist beispielsweise ein unter der Bezeichnung RPMI 1640 kommerziell erhältliches Medium, das nachstehend beschrieben ist, verwendbar. Weiterhin ist beispielsweise geeignet modifiziertes "Medium 199" (Haynes et al., 1976).The low molecular weight portions of serum and, if appropriate, culture media containing host cells, which are suitable for the cultivation of protozoa, are known. These are essentially amino acids, vitamins, peptone and physiologically acceptable salts. For Plasmodium falciparum, for example, a medium commercially available under the name RPMI 1640, which is described below, can be used. Also suitable is, for example, suitably modified "Medium 199" (Haynes et al., 1976).

Bestandteile solcher niedermolekularen Anteile von Nährmedien, die für die Kultivierung von Protozoen verwendet werden, sind beispielsweise :

Figure imgb0001
Figure imgb0002
dazu 5,94 g (25 mM) Hepes

  • 42 ml 5%ige NaHCO3-Lösung und
  • 10 ml Na-Glutaminat-Lösung (200 mmol).
Examples of such low molecular weight parts of nutrient media that are used for the cultivation of protozoa are:
Figure imgb0001
Figure imgb0002
 plus 5.94 g (25 mM) Hepes
  • 42 ml of 5% NaHCO 3 solution and
  • 10 ml Na glutaminate solution (200 mmol).

Als Serum wird bevorzugt eingesetzt: Humanserum A1, Rh ; Humanserum O.The preferred serum used is: human serum A 1 , Rh; Human serum O.

Als Wirtszellen werden verwendet:

  • Erythrozyten: für Plasmodien und Piroplasmen
  • Säuger- oder Hühnerfibroblasten: für Trypanosoma cruzi
  • Makrophagen: für Leishmania spp.
  • Lymphoblasten: für Theileria spp.
  • Insektenzellen: für Plasmodien
  • Karzinomzellen: für Toxoplasma grudii
The following are used as host cells:
  • Erythrocytes: for plasmodia and piroplasmas
  • Mammalian or chicken fibroblasts: for Trypanosoma cruzi
  • Macrophages: for Leishmania spp.
  • Lymphoblasts: for Theileria spp.
  • Insect cells: for plasmodia
  • Carcinoma cells: for Toxoplasma grudii

Falls Erythrozyten verwendet werden, sind brauchbar: Humanerythrozyten A, Rh ; Humanerythrozyten 0 .If erythrocytes are used, the following can be used: human erythrocytes A, Rh; Human erythrocytes 0.

Das folgende Beispiel soll die Erfindung erläutern:

  • In einem Fermenter wird eine sedimentierte Schicht gewaschener menschlicher Erythrozyten (A1, Rh-) mit einem definierten Medium (z.B. RPMI 1640) überschichtet. Die Menge des Mediums wird so berechnet, daß eine 1 - 10 %ige Erythrozytensuspension entsteht. Das Medium RPMI 1640 wird mit 5 - 10 % Humanserum der kompatiblen Gruppe substituiert. In den Fermenter wird eine Gasmischung bestehend aus 2 - 5 % CO2, 5 - 10 % 02 und 85 - 93 % N2 zur Oberflächenbelüftung eingeleitet. In einem kontinuierlichen Durchfluß strömt das Medium über die ruhende Schicht der Erythrozyten, nachdem es eine Dialysekammer (z.B. Amicon Hollow Fiber) durchlaufen hat. Auf der Gegenseite der Membran wird in einem zweiten Kreislaufsystem die 5 - 10fache Menge RPMI 1640 ohne Serum, Erythrozyten und Protozoen in entgegengesetzter Richtung gepumpt. Durch die Dialysemembran erfolgt der Austausch essentieller, niedermolekularer Nährsubstanzen, die infolge des Wachstums der Plasmodien und im geringeren Maße auch durch die Wirtszellen verbraucht werden, aus dem Vorratsgefäß in die Fermenterkultur sowie der Entzug von niedermolekularen inhibierenden Stoffwechselprodukten (z.B. Lactat) in das Außendialysat.
The following example is intended to explain the invention:
  • A sedimented layer of washed human erythrocytes (A 1 , Rh - ) is overlaid in a fermenter with a defined medium (eg RPMI 1640). The amount of the medium is calculated so that a 1-10% erythrocyte suspension is formed. The RPMI 1640 medium is substituted with 5-10% human serum from the compatible group. A gas mixture consisting of 2-5% CO 2 , 5-10% O 2 and 85-93% N 2 is introduced into the fermenter for surface aeration. The medium flows in a continuous flow over the resting layer of the erythrocytes after it has passed through a dialysis chamber (eg Amicon Hollow Fiber). On the opposite side of the membrane, 5 to 10 times the amount of RPMI 1640 without serum, erythrocytes and protozoa is pumped in the opposite direction in a second circulatory system. Through the dialysis membrane, essential, low-molecular nutrient substances, which are consumed due to the growth of the plasmodia and to a lesser extent also by the host cells, are exchanged from the storage vessel into the fermenter culture and the withdrawal of low-molecular-weight inhibiting metabolic products (e.g. lactate) into the external dialysate.

Die unter anderem durch den Parasitenstoffwechsel angereicherte Milchsäure (Lactat), die normalerweise eine Wachstums-inhibierende lokale pH-Änderung im Medium bedingt, wird durch die Dialysemethode entfernt, wodurch günstigere Wachstumsbedingungen erreicht werden (pH-Stabilität, Erhöhung des angebotenen Erythrozytenanteils und dadurch höhere Ausbeute an Malariaparasiten, Einsparung an unverbrauchten hochmolekularen Serumbestandteilen) :

Figure imgb0003
The lactic acid (lactate) enriched by the parasite metabolism, which normally causes a growth-inhibiting local pH change in the medium, is removed by the dialysis method, whereby more favorable growth conditions are achieved (pH stability, increase in the proportion of erythrocytes offered and thereby higher yield on malaria parasites, saving on unused high molecular serum components):
Figure imgb0003

Danach hergestellte Parasiten, vor allem Plasmodium falciparum, sind zur Herstellung eines antiparasitären (Malaria-) Impfstoffes geeignet. Die nach dem Wachstum geernteten Plasmodium falciparum Merozoiten (Plasmodien-Stadien in den Erythrozyten werden zum Beispiel durch Hämolyse aus der Wirtszelle befreit) werden ankonzentriert, attenuiert (chemisch oder physikalisch) und in eine Impfstoff gerechte Form gebracht.Parasites produced after this, especially Plasmodium falciparum, are suitable for the production of an anti-parasitic (malaria) vaccine. The Plasmodium falciparum merozoites harvested after growth (Plasmodia stages in the erythrocytes are freed from the host cell by hemolysis, for example) are concentrated, attenuated (chemically or physically) and brought into a vaccine-compatible form.

Gegenstand der Erfindung ist demnach insbesondere die Verwendung der nach dem erfindungsgemäßen Verfahren hergestellten Parasiten als wesentlicher Wirkstoff in einem Impfstoff oder in einem Diagnostikum.The invention accordingly relates in particular to the use of the parasites produced by the process according to the invention as an essential active ingredient in a vaccine or in a diagnostic agent.

Claims (2)

1. Verfahren zur Vermehrung von Protozoen in einer Suspension, welche Protozoen, Serum und gegebenenfalls Wirtszellen enthält, dadurch gekennzeichnet, daß die Suspension, getrennt durch eine Membran, die für niedermolekulare Stoffe durchlässig ist, mit einer Lösung von niedermolekularen Stoffen in Verbindung steht, die für die Vermehrung nötig sind, und daß diese Lösung inhibierende Stoffwechselprodukte aus der Suspension aufnimmt.1. A process for the propagation of protozoa in a suspension which contains protozoa, serum and optionally host cells, characterized in that the suspension, separated by a membrane which is permeable to low molecular weight substances, is connected to a solution of low molecular weight substances which are necessary for the multiplication, and that this solution absorbs inhibitory metabolites from the suspension. 2. Verwendung der nach dem Verfahren des Anspruchs 1 hergestellten Protozoen zur Herstellung eines Impfstoffes oder eines Diagnostikums.2. Use of the protozoa produced by the method of claim 1 for the production of a vaccine or a diagnostic.
EP81106855A 1980-09-09 1981-09-02 Process for the propagation of protozoal and their use Withdrawn EP0047485A3 (en)

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DE19803033776 DE3033776A1 (en) 1980-09-09 1980-09-09 PROTOCOL PROPOSITION METHOD
DE3033776 1980-09-09

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EP0047485A3 EP0047485A3 (en) 1982-12-08

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JP (1) JPS5783278A (en)
AR (1) AR226477A1 (en)
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DE (1) DE3033776A1 (en)
IL (1) IL63764A0 (en)
ZA (1) ZA816215B (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984002917A1 (en) * 1983-01-28 1984-08-02 Inst Medical W & E Hall EXPRESSION OF PLASMODIUM FALCIPARUM POLYPEPTIDES FROM CLONED cDNA
EP0166410A2 (en) * 1984-06-26 1986-01-02 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
GB2507944A (en) * 2012-08-14 2014-05-21 Jr Biomedical Ltd Apparatus for the culture of parasites

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, Band 66, Nr. 3, 1. August 1978, Seite 1484, Nr. 15111, Philadelphia, Pa (USA); F. NAKAMURA et al.: "Maintenenace of a certain rumen protozoal population in a continuous in vitro fermentation system"; & APPL. ENVIRON. MICROBIOL. 35(3), 500-506, 1978 *
BIORESEARCH INDEX, Band 74, 1974, Nr. 89690, Philadelphia, Pa (USA); A. BUAINAIN: "Studies on the cultivation of tzypanosoma-cruzi chagas 1909 in dialysis tubes in Warren medium and its use in the indirect immuno fluorescence reaction for the diagnosis of chagas disease"; & ILLUS. FACULTADE DE FARMACIA E ODONTOLOGIA DE ARARAQUARA, SAO PAULO BRAZIL, 1973 (RECD. 1974) 1-47 *
CHEMICAL ABSTRACTS, Band 93, Nr. 5, 4. August 1980, Seite 457, Nr. 41024z, Columbus Ohio (USA); A.H. FAIRLAMB et al.: "Trypanosoma brucei: maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis"; & EXP. PARASITOL. 1980, 49(3), 366-80 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984002917A1 (en) * 1983-01-28 1984-08-02 Inst Medical W & E Hall EXPRESSION OF PLASMODIUM FALCIPARUM POLYPEPTIDES FROM CLONED cDNA
GB2143830A (en) * 1983-01-28 1985-02-20 Inst Medical W & E Hall Expression of plasmodium falciparum polypeptides from cloned cdna
EP0166410A2 (en) * 1984-06-26 1986-01-02 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
EP0166410A3 (en) * 1984-06-26 1987-11-25 Us Commerce Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
GB2507944A (en) * 2012-08-14 2014-05-21 Jr Biomedical Ltd Apparatus for the culture of parasites
GB2507944B (en) * 2012-08-14 2019-11-13 Jr Biomedical Ltd A method of culturing plasmodium erythrocyte stage organisms

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IL63764A0 (en) 1981-12-31
JPS5783278A (en) 1982-05-25
ZA816215B (en) 1982-08-25
EP0047485A3 (en) 1982-12-08
DE3033776A1 (en) 1982-04-22
AR226477A1 (en) 1982-07-15

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